Properties of an ATP-Fueled, Cl--Dependent Proton Pump Localized in Membranes of Microsomal Vesicles from Maize Coleoptiles

Abstract

Abstract Vesicles prepared from the microsomal membrane fraction of maize coleoptiles possess an ATP-fueled H+-transport into the vesicles. It is highly possible that the vesicles from the microsomal membrane fraction actually are unchanged, native vesicles (originating from the ER or Golgi-apparatus) that can fuse with the plasmalemma or the vacuole. Therefore, they can reflect properties of the vacuole or the plasmalemma. The energy dependent acidification within the vesicles, which can be completely reverted through the addition of CCCP, was determined on the basis of photometric difference spectra using NR (Hager et al., Z. Naturforsch. 35 c, 794-804 1980). The proton pumps possess a very high substrate specificity; only ATP (+ Mg2+) can be used as substrate, while GTP, ITP, UTP and CTP or other nucleoside tri- or diphosphates cannot be used. DCCD and DES inhibit H+-ATPase completely, oligomycin has only a slight, orthovanadate no inhibitory effect at all. The energy dependent transport of H+ across the membrane takes place only in the presence of Cl- or Br- (not as well in the presence of I-). Other anions (F-, NO- 3, SO2- 4, SCN-, IDA-, H2BO- 3 cannot cause an intravesicular acidification through ATP if chloride (or Br-) is not present. In the presence of chloride, however, some of these anions inibit the H+/Cl--symport (J-, NO- 3, SO2- 4, SCN-, H2BO- 3). They obviously are in competitive interaction with Cl- ions for Cl--binding sites on a carrier or channel without being able to be transported themselves. Pi, which renders the acidification of the vesicles at a low rate possible without Cl-, is the only tested anion which can augment the Cl- dependent acidification. This supports the idea that either Pi functions as a positive effector on the Cl-transport or that a Cl-/Pi-antiporter exists which reduces Cl- accumulation and therefore facilitates the Cl- coupled H+ transport into the vesicles also. The anion transport inhibitor, DIDS, blocks the ATP-dependent H+-transport. This again supports the idea of a relatively tight coupling between H+ and Cl- transport in a possibly electroneutral system. The presence of monovalent cations, such as K+, Na+, Li+ and choline+, are not important for H+ transport. The dependence of the ATP-fueled acidification of the vesicles on the same anion pattern which seems to regulate elongation growth and stomata aperture speaks for the eminent importance of the H+-pump and vesicles described in this report for growth and turgor of plant cells.